You are on page 1of 5


AAnn IInnddeeppeArticle
ennddeenntt,, OOppeenn AAcccceessss,, PPeeeerr RReevviieewweedd,, NNoonn--PPrrooffiittDeepa
all al. (2012)

International Journal of Life Sciences

Yeeaarr 22001122 V
mee 66,, IIssssuuee 11

Research Article

Antioxidant and calcium levels in mature and immature diabetic

cataract lens
Deepa K 1, *, Manjunatha Goud B.K 2, Suma M.N. 1, Devaki R.N. 1 , Nandini M. 3 and Sudhir 4
Department of Biochemistry, JSS Medical College, JSS University, Mysore, India. 2 Department of Biochemistry, Ras AI Khaimah Medical
and Health Sciences University, UAE. 3 Department of Biochemistry, Kasturba Medical College, Mangalore, India. 4 Department of Community
Medicine, JSS Medical College, JSS University, Mysore, India.

Corresponding author ABSTRACT

Email: Cataract is considered a major cause of visual impairment in diabetic patients as the incidence and progression of cataract is elevated in patients with diabetes mellitus. The pathogenesis of diabetic
cataract development is still not fully understood. Due to increasing numbers of type 1 and type 2
diabetics worldwide, the incidence of diabetic cataracts steadily rises. Even though cataract
Phone No:
surgery, the most common ophthalmic procedure worldwide, is an effective cure, the elucidation of
+91 - 9900595772
patho- mechanisms to delay or prevent the development of cataract in diabetic patients remains a
challenge. Furthermore, patients with diabetes mellitus have higher complication rates from
Fax No: cataract surgery. Both diabetes and cataract pose an enormous health and economic burden,
-- particularly in developing countries, where diabetes treatment is insufficient and cataract surgery
often inaccessible. Hence the objective of the study was to assess the antioxidant levels and
Editor calcium levels in different stages of cataract. Our study showed a significant reduction in lens
Ganesh Kumar Agrawal antioxidant activity with significantly increased calcium levels in mature stage of cataract,
suggesting decreased antioxidant activity plays an important role in diabetic cataract by causing
DOI deposition of calcium in lens.
Key words: Diabetes; Cataract; Antioxidant; Calcium

INTRODUCTION Diabetic cataract is more prevalent in diabetic

adults compared to non diabetics. It depends on
Worldwide more than 285 million people are duration of diabetes and glycemic status. It
affected by diabetes mellitus. This number is expected progresses rapidly if the blood sugar levels are not
to increase to 439 million by 2030 according to the maintained within normal range. The mechanism
International Diabetes Federation. behind the diabetic cataract is mainly due to
hyperglycemia induced oxidative stress. Excessive
Diabetes mellitus is characterized by absolute levels of glucose reaching the mitochondria leads to
or relative deficiencies in insulin secretion and/or an overdrive of electron transport chain, resulting in
insulin action associated with chronic hyperglycemia over production of superoxide anions, normally
and disturbances of carbohydrate, lipid and protein scavenged by mitochondrial superoxide dismutase.
metabolism. During diabetes, persistent When latter fails oxidative stress develops and it is
hyperglycemia causes increased production of free proposed that this mechanism is responsible for
radicals especially reactive oxygen species (ROS), for activation of all major pathways responsible for
all tissues from glucose auto-oxidation and protein diabetic complications of which includes, activation of
glycosylation (Aragno et al., 1999). Free radicals are sorbitol pathway, glycation and protein kinase
generated as by-products of normal cellular activation (Brownlee, 2000).
metabolism; however, several conditions are known
to disturb the balance between ROS production and Hyperglycemia can also stimulate oxidative
cellular defense mechanisms (Bonnefont et al., 2000). stress by the auto oxidation of glucose in the presence

56 | International Journal of Life Sciences • ISSN 2091–0525 • Year 2012 • Volume 6 • Issue 1
Research Article Deepa et al. (2012)

of transition metals as well as generation of ROS history of steroid intake, other ophthalmic disease,
during the process of glycation. Oxidative stress also and systemic disorders were excluded from the study.
affects the expression of antioxidant enzymes by its The study was approved by the Institutional Time
action on signal transduction. Hyperglycemia can also Bound Research Committee. A written informed
cause inactivation of the existing enzymes by consent was taken from the subjects. Postoperatively
glycation. Thus oxidative stress may develop by cataractous lens were collected in ice cold 0.9%
insufficient antioxidant activity even if ROS Normal saline container and transferred in ice box to
production is within physiological range (King et al., the place of analysis and analyzed on the same day
1996) immediately for the following parameters: For
determining the antioxidant activity and calcium
Calcium is an important extracellular cation in levels in lens, the lens was homogenized with 1.2ml
the body, total calcium in the body is about 1-1.5kg. ice cold 0.01M phosphate buffer pH 7.4. Mixture was
99% is in bone and 1% in extracellular fluid. It is centrifuged at 2500rpm for 10mins. 100µl
present both in extracellular and intracellular supernatant was used for analysis. The total lens
compartments. The cell membrane is generally antioxidant activity was determined by Koracevic’s
impermeable to calcium ions. Normally the calcium method (Koracevic et al., 2001) the assay measures
levels are maintained at low concentration inside the the capacity of antioxidant to inhibit the production of
cell. This is maintained by Na+ K+ exchanger; Na+ Ca2+ thiobarbituric acid reactive substances from sodium
exchanger and calcium ATPase pump (Galvan, 1988). benzoate under the influence of the oxygen free
Optimum intracellular calcium is necessary to radicals derived from Fenton reaction. The reaction
maintain lens transparency. Calcium is maintained in was measured spectrophotometrically at 535nm.
micromoles range in the cytoplasm by regulatory Calcium in the lens was determined by
systems. In normal lens, the ionized calcium is less Cresolphthalein complexone method (Stern, 1957)
than 1% of the total concentration and below 0.1% of using calcium kits from Aspen Lab Pvt Ltd.
the level in surrounding aqueous humor (Tomlinson Cresolphthalein complexone a metal complex dye
et al., 1991). reacts with calcium ions in alkaline medium forming a
purple color. Intensity of the color formed is directly
Metal ions are known to play an essential role
proportional to the calcium concentration and was
in living systems, both in growth and in metabolism.
measured photometrically at 570nm. Since we
Impaired metabolism of trace elements is observed in
analyzed in lens tissue we need to express the levels
diabetic patients. Alteration in the capacity to
of total antioxidant activity and calcium in milligrams
maintain normal calcium homeostasis have been
of proteins .So protein content was determined by the
suggested to underlie the reduced cellular functions
Lowry’s method (Lowry et al., 1951) blue color
characteristic of aging process and predisposes the
obtained was measured spectrophotometrically using
senescent organism to diverse pathologies viz
Bovine albumin as standard
Cancer, Heart disease, Neurodegenerative disease
including Cataract (Thomas et al., 2000; Ernesto et Statistical Analysis
al., 1987) Altered calcium homeostasis due to
oxidative stress leads to increased intracellular The data was statistical analysis by epi-info
calcium levels and in turn leads to cataract formation software using Analysis of Variance [ANOVA]. Since
(Wride, 1996; Nakamura et al., 2000) two way ANOVA showed that the interaction between
cataract and maturity is significant, the comparison
The aim of the study was to evaluate the total
between the type of cataract and maturity was done
antioxidant activity and calcium levels in diabetic
separately using independent ‘t’ test. The results were
patients lens depending on type of cataract and to
expressed as Mean ± Standard deviation.
correlate the levels of calcium with the antioxidant
activity in progression of cataract formation.
The significance level at p<0.05 was
MATERIALS AND METHODS considered statistically significant. To correlate the
levels of calcium with the antioxidant activity,
The study included fifty diabetic subjects Pearson’s correlation co-efficient was worked out.
having cataract, aged between 45 -70 years of either
sex. Later they were grouped into group 1- immature
diabetic cataract patients and group 2- mature
diabetic cataract patients based on ophthalmoscopic
examination by ophthalmologist. Patients with
57 | International Journal of Life Sciences • ISSN 2091–0525 • Year 2012 • Volume 6 • Issue 1
Research Article Deepa et al. (2012)

Table 1. Comparison of parameters between immature and mature cataract.

N Immature N Mature
Dry weight of lens 24 56±1.79 26 70±2.57**
Total Proteins 57±0.56 65±0.66*
Antioxidant 1.320± 0.208 0.820± 0.085***
(mg/mg of protein)
Calcium 24 0.805± 0.416 26 1.024± 0.056***
(mg/mgof protein)

RESULTS number of studies have focused on the individual

antioxidants. A decreased level of erythrocyte
The comparison of the antioxidant activity glutathione peroxidase, glutathione reductase has
and calcium levels between mature and immature been reported in diabetic cataract. Likewise decrease
cataractous lens are presented in Table 1. A levels of SOD, Catalase, GSH; Glutathione peroxidase
significant reduction in the Antioxidant activity in has also been reported in lens (Nishikawa et al., 2000;
mature cataractous lens was observed with the mean Chandrasena et al., 2006). The role of calcium in
lenticular metabolism has been studied by many
value of 0.82 mg/mg of proteins against the value of
workers in animal models and their study results are
1.32mg/mg of protein seen in immature lens. A as follows:
significant increase in the lens calcium was observed
in mature cataract. The mean value being P.G. Biju (Biju et al., 2007) and Lifei Wang
1.024mg/mg of protein against 0.805mg/mg of (Wang et al., 2001) studies showed that the increased
lenticular calcium is due to altered calcium ATPase
protein observed in immature cataract. A negative
Pump activity due to oxidative stress, which in turn
correlation was seen between lens antioxidant leads to activation of calpain proteases which is
activity and lens calcium and r value was -0.966. responsible for proteolysis. In Fein et al. studies,
increased lenticular calcium causes aggregation of
DISCUSSION high molecular weight proteins (Fein et al., 1979).In
G-Duncan et al, KR Hightower et al, (Duncan et al.,
Dysfunctional neuro-endocrine–endocrine 1993; Hightower et al., 1980) studies the altered
interactions contribute to the disturbances in trace calcium homeostasis is due to altered calcium ATPase
element metabolism and cause severe complications and sodium calcium exchange mechanism and
in diabetes mellitus (Tallman et al., 1999). The thereby affecting calcium transport across lens.
present results showed that the levels of calcium Investigations by Tang (Tang et al., 2003) have shown
were statistically increased in the mature cataractous that the traces of calcium in lens are in some way
lens compared with immature diabetic cataract related to the maintaince of normal permeability and
patients (p<0.001), and the antioxidant activity is regulation of dynamic equilibrium between ionic
statistically decreased in mature diabetic cataractous constituents of its lens and its surrounding fluid.
lens (p<0.001).
Thus there is a significant increase in calcium
Aksoy et al. (2001) have reported, a reduced content with the onset of maturity is due significantly
total antioxidant status in aqueous humor in patients decreased antioxidant levels. Altered calcium
with diabetic cataract than those with senile cataract homeostasis, due to oxidation of the regulatory
(Aksoy et al., 2001). A much reduced antioxidant proteins or by damage to the plasma membrane by
activity in diabetic patients could be explained by lipid peroxidation, which thereby affects the
hyperglycemia induced complication such as reduced membrane functions and structure, may be the cause
gene expression of antioxidant enzymes and inactivity for increase in calcium levels in these patients. In lens,
of existing enzymes by glycation (King et al., 1996). calcium is essential for various lens fiber cell
Thus resulting, overall reduction in antioxidant levels processes including its differentiation. An optimum
which fails to defend oxidative stress, there by concentration is essential for activation of Ca-ATPase
accelerating the process of cataractogenesis. A pump. Altered calcium homeostasis due to oxidative
58 | International Journal of Life Sciences • ISSN 2091–0525 • Year 2012 • Volume 6 • Issue 1
Research Article Deepa et al. (2012)

Figure 1. Comparison of lens antioxidant activity between Immature and Mature diabetic cataract patients

Figure 2. Comparison of lens calcium levels in Immature and Mature Diabetic Cataract patients

stress leads to increased intracellular calcium levels status which is responsible for opacification of lens
and in turn leads to cataract formation by following and also calcium plays an important role in causation
mechanisms, increased intracellular calcium interacts of diabetic cataract.
with cytoplasmic proteins causing protein oxidation
and aggregation of high molecular weight proteins. REFERENCES
This leads to increase in soluble protein aggregates.
Elevated calcium levels leads to widespread Aragno, M.E; Tamagno, V.; Gatto, E.; Brignardello, S.; Parola,
O.; Danni, G. & Boccuzzi. 1999. Dehydroepiandrosterone
activation of proteases particularly calpain, leading to protects tissues of streptozotocin-treated rats against oxidative
proteolysis of crystallin. Increased calcium reduces stress. Free Radical Biology and Medicine. 26(11/12): 1467–
the calcium ATPase pump activity and sodium 1474.
potassium ATPase activity thereby altering the
Biju, P.G.; Rooban, B.N.; Lija, Y.; Gayathri devi, V.;
membrane permeability of lens (Thomas et al., 2003) Sahasranamam, V. & Abraham, A. 2007. Drevogenin – D
prevents selinite – induced oxidative stress and Calpain activity in
The present data revealed that hyperglycemia cultured rat lens. Molecular vision. 13:1121-1129.
produced marked oxidant impact as evident by the
significant decrease in antioxidants status in lens. Bonnefont rousselot, D.; Bastard, J.P.; Jaudon, M.C. & Delattre,
J. 2000. Consequences of the diabetic status on the
This definitely proves that oxidants play an important
oxidant/antioxidant balance. Diabetes and Metabolism. 26:
role as well in genesis of cataract. Hence, estimation 163–176.
of total antioxidant activity can serve as good
predictive marker in assessing the disturbed redox
59 | International Journal of Life Sciences • ISSN 2091–0525 • Year 2012 • Volume 6 • Issue 1
Research Article Deepa et al. (2012)

Brownlee, M. 2000. Negative consequence of glycation. mellitus type 2. Canadian Journal of Physiology 77:919–33.
Metabolism. 49(2): 9-13. PMid:10606438

Carafoli, E. 1987. Intracellular calcium homeostasis .Annual Tang, D.; Borchman, D.; Yappert, M.C.; Vrensen, G.F. & Rasi, V.
Review of Biochemistry 56:395-434. 2003. Influence of Age, Diabetes and Cataract on calcium, lipid
10.1146/ PMid:3304139 calcium and protein calcium relationships in human lenses.
Investigative Ophthalmology and Visual sciences 44(5):2059-
Chandrasena, L.G.; Chackrewarthy, V.; Teckla, P.; Perera, M.J. 66.
& de silva, D. 2006. Erythrocyte Antioxidant Enzymes in Patients
with cataract. Annals of clinical & laboratory science 36;201- Thomas, C.; Squier & Diana J. 2000. Protein oxidation and age
204 dependent alteration in calcium homeostasis. Frontiers in
Bioscience 5:504-526.
Duncan, G.; Webb, S.F.; Dawson, A.P.; Bootman, M.D. & Elliott, PMid:10799358
A.S. 1993. Calcium regulation in tissue cultured human and
Bovine lens epithelium cells. Investigative Ophthalmology and Tomlinson, J.; Bannister, S.C.; Croghan, P.C & Duncan, G. 1991.
Visual sciences 34.2835-2845 Analysis of rat lens calcium fluxes: evidence for Na- Ca exchange.
Experimental eye Research 52(5):619-627.
Fein, T.; Pande, A. & Spector, A. 1979. Further investigation of 10.1016/0014-4835(91)90065-M
role of calcium in human lens protein, aggregation. Investigative
Ophthalmology and Visual sciences 18(7):761-765. Wang, L.; Christensen, B.N.; Bhatnagar, A. & Srivastava, S.K.
2001.Role of Calcium dependent protease in globulisation of
Galvan, A. 1988. Calcium regulation by lens plasma membrane isolation of isolated rat lens cortical fibers cells. Investigative
vesicles. Archives of Biochemistry and Biophysics 264(2): 472- Ophthalmology and Visual sciences 42:194-99

Hightower, K.R.; Leverenz, V. & Reddy, V.N. 1980. Calcium

transport in lens. Investigative Ophthalmology and Visual
sciences. 19(9): 1059-1065
Submit your next manuscript to IJLS
with a -
Hulya; A.; Keles, S.; Kocer, I.; Akcay, F.C. 2001. Diabetic cataract i) Convenient online submission,
and the total antioxidant status in Aqueous Humor. Clinical ii) Rapid editorial review followed by peer review,
chemistry and laboratory medicine 39(2):143-145 iii) No space constraints or color figure charges,
iv) Immediate publication on acceptance.
King, G.L.; Kunisaki, M.; Nishio, Y.; Inoguchi, T. & Shiba, T.
1996. Biochemical and molecular mechanisms in the
development of diabetic vascular complications. Diabetes Note1 - Authors/contributors are responsible for originality,
45:105-108. contents, correct references, and ethical issues.

Koracevic, D.; Koracevic, G.; Djordjevic, V.; Andrejevic, S. & Note2 - Authors/contributors are required to write their text in
Cosic, V. 2001. Methods of the measurement of antioxidant good English (American or British usage but not a mixture of
activity in human fluids. Journal of Clinical Pathology. 54:356- these), and authors who need assistance with language editing
361 PMid: and copyediting services pre-/post-submission please visit
11328833 PMCid:1731414 for more information.

Lowry, O.H.; Rosebrough, N.J.; Farr, A.L. & Randall, R.J. 1951. © International Journal of Life Sciences (IJLS)
Protein measurement with Folin- Phenol reagent. Journal of
Biological Chemistry. 193: 265-270

Wride, M.A. 1996. Cellular and molecular features of lens

differentiation: a review of recent advances. Differentiation.
61(2) : 77-93

Nakamura, Y.; Fukiage, C.; & Shih, M. 2000. Contribution of

Calpain LP 82 induced proteolysis to experimental
cataractogenesis in mice. Investigative Ophthalmology and
visual Sciences 41:1460-66.

Nishikawa, T.; Edelestein, D.; Du, X.L. & Yamagishi, S. 2000.

Normalizing mitochondrial superoxide production blocks three
pathways of hyperglycemic damage. Nature. 404:787-790. PMid:10783895

Stern, J & Lewis, W.H.P. 1957. The colorimetric estimation of

calcium in serum with O-Cresolpthalein complexone. Clinica
Chemica Acta 2(6) 576-580.

Tallman, D.L. & Taylor, C.G. 1999. Potential interactions of zinc

in the neuroendocrine – endocrine disturbances of diabetes

60 | International Journal of Life Sciences • ISSN 2091–0525 • Year 2012 • Volume 6 • Issue 1